Breast cancer brain metastasis (BCBM) is a devastating disease whose mechanisms are understudied and remain largely unknown. For example, while human epidermal growth factor receptor1 and 2 (EGFR and HER2) are high-risk predictors of BCBM, HER2/EGFR-targeted therapies have proven to be mostly ineffective against BCBM. Although notions that circulating tumor cells (CTCs) acting as seeds of intractable metastasis are established, virtually nothing is known about the properties and biomarkers of breast cancer brain-colonizing CTCs. Therefore, there is an urgent need for new CTC target biomarkers to combat BCBM. We have discovered CTCs that do not express the epithelial cell adhesion molecule (EpCAM-negative CTCs) and possess high BCBM competency in xenografts. These CTCs could not be detected by CellSearchTM, a FDA-cleared prognostic test which evaluates only EpCAM-positive CTCs. We found two promising markers in EpCAM-negative CTCs to predict BCBM in the clinic: Notch1, an important tumor-initiating cell biomarker, and heparanase (HPSE), a potent tumorigenic, angiogenic and pro-metastatic molecule. Our results indicate that BCBM-inducing CTCs have a Brain Metastasis-Selected Marker (BMSM) profile or HER2+/EGFR+/Notch1+/HPSE+ CTCs. Notch1 and HPSE can thus represent novel and specific CTC biomarkers. Furthermore, we detected highest expression of the tumor modulator Merlin (Neurofibromatosis type2) in Notch1/HPSE-expressing CTCs. The phosphorylation of Merlin (P-S518 Merlin) by PAK1 kinase resulted in oncogenic functions of Merlin which associate with BMSM CTC pathways and their regulation. Based on these findings, our hypothesis is that Notch1 and HPSE are novel CTC biomarkers that can predict the presence of primary breast cancer brain metastasis; and they can be potential therapeutic targets to prevent secondary BCBM. Objective of this proposal is to perform preclinical translational research and target validation to nominate HPSE and Notch1 as critical biomarkers of breast cancer brain-homing CTCs. We will study CTCs from HER2+ patients since this breast cancer subtype has a much higher than average risk of developing BCBM. Specifically, we will: 1) determine effects of therapeutic inhibition and regulation of Notch1 and HPSE CTC markers on BCBM onset; 2) link combinatorial Notch1/HPSE CTC subsets to clinical BCBM; 3) define roles of Merlin affecting BMSM CTC pathways and BCBM oncogenesis. This inter-institutional project by an inter-disciplinary and well-integrated team will study and validate new and specific CTC markers responsible for CTC-induced BCBM. This project is paradigm-shifting and has high therapeutic impact. We are uniquely positioned to perform this study not only for having access to an extensive cohort of blood samples and volume from patients diagnosed with or without BCBM but also for the availability of human CTC lines our laboratory was first to develop, and the extensive expertise combining multiple and complementary CTC platforms and approaches unlike other groups in the country.